【摘要】：目的建立體外BMSCs、NPCs非接觸式共培養(yǎng)體系,探討不同條件下NPCs誘導BMSCs向類髓核細胞分化的效果及影響因素。方法1.取新西蘭大白兔4只,采用改良全骨髓細胞貼壁法獲取BMSCs,進行BMSCs體外培養(yǎng)、純化、擴增；2.通過細胞形態(tài)學觀察、繪制細胞生長曲線、流式細胞儀檢測,觀察BMSCs的生物學性狀并對其進行鑒定：3.取新西蘭大白兔4只,采用改良酶聯(lián)消化法獲取NPCs,體外細胞培養(yǎng)技術對NPCs進行原代、傳代培養(yǎng)；4.通過細胞形態(tài)學觀察、繪制細胞生長曲線、甲苯胺藍染色、免疫組織化學染色、RT-PCR檢測,觀察NPCs的生物學性狀并對其進行鑒定；5.取培養(yǎng)鑒定的BMSCs、NPCs,根據(jù)BMSCs與NPCs不同的細胞比例分為共培養(yǎng)組(2：1)、共培養(yǎng)組(1：1)、共培養(yǎng)組(1：2)、共培養(yǎng)組(1：4)、共培養(yǎng)(1：1)+TGF-β1組,以TGF-β1誘導組作為對照,建立BMSCs、NPCs非接觸式共培養(yǎng)體系。各組培養(yǎng)15天后對經誘導的BMSCs進行實時熒光定量PCR檢測,通過檢測Aggrecan、 Collagen Ⅱ基因的相對表達量,從基因水平觀察誘導效果,并對結果進行分析。結果 1.采用改良全骨髓細胞貼壁法能夠獲取BMSCs,經流式細胞儀檢測純度較高；2.采用改良酶聯(lián)消化法能夠獲取NPCs,經基因水平、蛋白水平鑒定,均有Aggrecan、Collagen Ⅱ表達；3.不同條件下BMSCs、NPCs非接觸式共培養(yǎng)體系和TGF-β1誘導體系均可誘導BMSCs向類髓核細胞分化,實時熒光定量PCR檢測Aggrecan、Collagen Ⅱ基因表達陽性；4.不同細胞比例的共培養(yǎng)體系下,較高NPCs比例的共培養(yǎng)組與較低NPCs比例的共培養(yǎng)組相比,其誘導的類髓核細胞Aggrecan、Collagen Ⅱ基因表達量更高,組間差異有統(tǒng)計學意義；5.共培養(yǎng)條件下,經1：1細胞比例+10ng/ml TGF-β1誘導后類髓核細胞Aggrecan基因表達量較1：4比例共培養(yǎng)組的更高,差異有統(tǒng)計學意義。Collagen Ⅱ基因表達量與1：4比例共培養(yǎng)組接近,差異無統(tǒng)計學意義。6.體外TGF-β1誘導BMSCs向類髓核細胞分化后,類髓核細胞Aggreca、 Collagen Ⅱ基因表達量與細胞比例1：2共培養(yǎng)條件下誘導的類髓核細胞基因表達量相近,差異無統(tǒng)計學意義；結論體外TGF-β1共培養(yǎng)條件下NPCs均可誘導BMSCs向類髓核細胞分化,體系中BMSCs/NPCs的比例、TGF-β1含量對誘導效果均起重要作用。
[Abstract]:Objective to establish a non-contact co-culture system of BMSCs,NPCs in vitro and to investigate the effect of NPCs on the differentiation of BMSCs into nucleus pulposus cells under different conditions. Method 1. BMSCs, was obtained from 4 New Zealand white rabbits by modified whole-bone marrow cell adherence method. BMSCs was cultured, purified and amplified in vitro. 2. The biological characters of BMSCs were observed and identified by cell morphology observation, cell growth curve and flow cytometry. NPCs, cells were obtained from 4 New Zealand white rabbits by modified enzyme-linked digestion (Elisa) and NPCs was subcultured in vitro. 4. Cell growth curve, toluidine blue staining, immunohistochemical staining and RT-PCR detection were used to observe and identify the biological characteristics of NPCs. According to the ratio of BMSCs and NPCs, BMSCs,NPCs, was divided into three groups: co culture group (2:1), co-culture group (1:1), co-culture group (1:2), co-culture group (#timea-) TGF- 尾 1 group. BMSCs,NPCs non-contact co-culture system was established with TGF- 尾 1 induction group as control. After 15 days of culture, the induced BMSCs was detected by real-time fluorescence quantitative PCR. The relative expression of Aggrecan, Collagen 鈪，

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